Manipulation of viral protein production using the PCNA of halovirus фCh1 via alternative start codon usage

Abstract

• Proliferating Cell Nuclear Antigen (PCNA) is a scaffold protein principally found at the centre of replication, coordinating with a wide array of interaction partners. фCh1, unusually for a virus, encodes a putative PCNA. • Natrialba magadii, the only known host of фCh1, also encodes a putative PCNA of high sequence similarity, differing majorly in the presence of an alternate GTG start codon in the фCh1 variant 5’ of the ATG start codon resulting in a larger protein. Homologous recombination was used to delete the viral PCNA gene. • Complementation and overexpression strains with plasmid expressed viral PCNA variants were created and analyzed for growth and lytic behavior, viral protein levels, and virus titer. • Deletion of фCh1-ORF59, encoding PCNA ΦCh1 resulted in a significant reduction in the virus titer, reduced viral protein load, and reduced lysis. • Complementation and overexpression with WT, and mutant variants of ORF59revealed that the 5’ GTG to ATG sequence enables фCh1 to control its equilibrium with the host. The different variants have a broad range of variable effects on each of the phenotypes observed. • Experiments with a halophilic beta-galactosidase assay revealed a cis/trans mediated interaction between the viral PCNA and the origin of replication of φCh1 modulated by the 5’ GTG to ATG sequence. The virally encoded PCNA is not essential, but is crucial, to the behavior of the virus фCh1. The 5’ region upstream of the primary ATG codon and its regulation with an alternate start codon is essential to the homeostasis of the virus-host relationship. Proliferating Cell Nuclear Antigen (PCNA) is a scaffold protein principally found at the centre of replication, coordinating with a wide array of interaction partners. фCh1, unusually for a virus, encodes a putative PCNA. Natrialba magadii, the only known host of фCh1, also encodes a putative PCNA of high sequence similarity, differing in the presence of an alternate GUG start codon 5’ of the AUG start codon resulting in a larger protein. Homologous recombination was used to delete the viral PCNA gene. Complementation and overexpression strains with plasmid expressed viral PCNA variants were created and analyzed for growth and lytic behaviour, viral protein levels, and virus titer. Deletion of фCh1 ORF59, encoding PCNA ФCh1 resulted in a significant reduction in the virus titer, reduced viral protein load, and reduced lysis. Complementation and overexpression with WT and mutant variants of ORF59 revealed that modification of the GTG start codon to ATG changes the life cycle in terms of production of progeny virus particles. The different variants have a broad range of variable effects on each of the phenotypes observed. Experiments with a halophilic beta-galactosidase assay revealed a cis/trans mediated interaction between the viral PCNA and the origin of replication of фCh1 modulated by the 5’GTG to ATG sequence. The virally encoded PCNA is not essential, but is crucial, to the life cycle of the virus фCh1. The 5’ region upstream of the primary AUG codon and its regulation with an alternate start codon is essential to the homeostasis of the virus-host relationship.